Moreover, treatment of bL for 15 and 30 min did not affect SIRT1 localization (Physique 3B). chambers, ALDEFLUOR assay, and mammosphere formation assay. Here, we show that bL inhibited the proliferative ability of mammospheres derived from BCSC marker-positive cells, MDA-MB-231, in an NQO1-dependent manner. The bL treatment efficiently downregulated the expression level of BCSC markers cluster of differentiation 44 (CD44), aldehyde dehydrogenase 1 family member A1 (ALDH1A1), and discs large (DLG)-associated protein 5 (DLGAP5) that was recently identified as a stem-cell proliferation marker in both cultured cells and mammosphered cells. Moreover, bL efficiently downregulated cell proliferation and migration activities. These results strongly suggest that bL could be a therapeutic agent for targeting breast-cancer stem-cells with proper NQO1 expression. = 3; ** < 0.01, *** < 0.001. (E) Cell lysates obtained from MCF7 and MDA-MB-231 cells were subjected to Western blot analysis to measure the protein expression level of BCSC markers determined by quantitative RT-PCR; -actin was used as a loading control. 2.2. -Lapachone-Mediated NQO1 Activation Regulates DLGAP5 and CD44 Expression Levels To gain insight into the possible mechanism via which NQO1 regulates DLGAP5 and CD44 expression, we created MDA-MB-231 cells stably expressing either NQO1 (NQO1 stable cells) or the vector control (control cells). The expression of each gene was compared in control cells and in two different clones of NQO1 stable cell lines with or without bL. Interestingly, the gene expression Biochanin A (4-Methylgenistein) of DLGAP5 and CD44 was downregulated by bL treatment in the presence of NQO1 in MDA-MB-231 cells, but not in control cells, indicating that NQO1 is required for the bL-mediated downregulation of these genes (Physique 2A,B). In contrast, the ALDH1A1 expression level was not altered by bL treatment regardless of NQO1 expression in both control and NQO1 stable cell lines (Physique 2C). To verify the effect of bL-mediated NQO1 on protein expression, Western blot analysis was performed after bL treatment on control and NQO1 stable cells (Physique 2D). As expected, bL treatment did not affect the protein expression levels of DLGAP5, CD44, or ALDH1A1 in control cells. Interestingly, the DLGAP5 protein level was increased Biochanin A (4-Methylgenistein) by NQO1 expression alone, but bL treatment dramatically decreased the DLGAP5 protein expression in NQO1 stable cells. Moreover, Biochanin A (4-Methylgenistein) CD44 expression was not affected by NQO1 expression alone, but was also decreased by bL treatment in NQO1 stable cells. These results imply that DLGAP5 is usually upregulated by NQO1 alone via an unknown mechanism, and that bL is essential for NQO1-mediated downregulation of both DLGAP5 and CD44 gene and protein expression. Unexpectedly, ALDH1A1 was also downregulated by bL treatment in NQO1 stable cells, which was different from the result shown in the mRNA expression pattern (Physique 2C), suggesting that NQO1 activation by bL might regulate ALDH1A1 expression at the post-translational modification level (Physique 2D). Open in a separate window Physique Rabbit Polyclonal to BRCA1 (phospho-Ser1457) 2 The -lapachone (bL) compound suppresses the expression of BCSC markers in an NQO1-dependent manner. (ACC) The mRNA expression levels of DLGAP5, CD44, and ALDH1A1 were compared among MDA-MB-231 and two impartial clones of NQO1 stable cells (NQO1 #1 and #2) with or without bL (2 M) over a 24-h treatment. was used as an internal control, and each expression level was normalized to that of = 3; * < 0.05, ** < 0.01. (D) Protein expression levels of DLGAP, CD44, and ALDH1A1 were compared between MDA-MB-231 and two impartial clones of NQO1 stable cells (NQO1 #1 and #2) with or without bL (2 M) for a 24-h treatment; -actin was used as a loading control. 2.3. Sirtuin 1 (SIRT1) Is Not Involved in bL-NQO1-Mediated Biochanin A (4-Methylgenistein) Gene Expression and Cell Death SIRT1 is an NAD+-dependent deacetylase and regulates gene expression by regulating acetylation on proteins . Because SIRT1 is usually observed in both the cytosol and nucleus, its localization is regarded as an important event in the regulation of cell proliferation . In addition, NQO1 activated by bL accelerates the conversion of NADH to NAD+, and increased cellular NAD+ levels may affect cancer cell proliferation. Therefore, we hypothesized that a cellular NAD+ level increased by bL-NQO1 may activate SIRT1 and regulate BCSC marker gene expression. To verify our hypothesis, we firstly examined SIRT1s cellular localization after bL treatment. We fractionated NQO1 stable cells after treatment with bL for 24 h. NQO1 was observed mainly in the cytoplasmic fraction, and DLGAP5 and ALDH1A1 were observed in the nucleus. Notably, the DLGAP5 and ALDH1A1 protein levels were again downregulated by bL treatment in the presence of NQO1 expression (Physique 3A). However, Biochanin A (4-Methylgenistein) we could not find any difference in SIRT1 protein amount in the cytoplasmic and nuclear fractions by bL treatment. Moreover, treatment of bL for 15 and 30 min did not affect SIRT1 localization (Physique 3B). Finally, we performed assessments with an SIRT1 inhibitor to see whether SIRT1 is usually involved in bL-NQO1-mediated.